KR20090056171A - Anti-inflammatory composition containing geranium nepalense extract as an effective factor - Google Patents

Abstract

A Geranium nepalense sub sp. thunbergii extract having anti-inflammatory effect through inhibition of NF-kappaB, cyclooxygenase-2, and chondroitinase is provided to prevent the chronic inflammation without side effect and durability. A Geranium nepalense sub sp. thunbergii extract having anti-inflammatory effect is isolated through the hot water extraction of Geranium nepalense sub sp. thunbergii or methanol extraction using a methanol. A pharmaceutical composition for anti-inflammation comprises the Geranium nepalense sub sp. thunbergii extract. A fermented milk for anti-inflammtion containing the Geranium nepalense sub sp. thunbergii extract is manufactured by inoculating HY2782(Lactobacillus casei HY2782)(deposit number KFCC-10813); resolving the hot water extract of fruit juice concentrate, dietary fiber, glucose, oligosaccharide, calcium, vitamin, and Geranium nepalense sub sp. thunbergii; mixing; and stirring.

Description

Anti-inflammatory composition containing Geranium nepalense extract as an effective factor

The present invention relates to a heterogeneous grass extract having an anti-inflammatory effect and a composition containing the same as an active ingredient, and more particularly, the present invention relates to NF-κB, cyclooxygenase-2, and chondroitinase ( The present invention relates to an extract of erythrocytes having anti-inflammatory efficacy through the inhibition of activation of chondroitinase and an anti-inflammatory composition, fermented milk, beverage and health functional food containing the same as an active ingredient.

Inflammatory reactions are biological defense reactions for repairing and repairing damage caused by invasion that causes certain organic changes in cells or tissues. Local blood vessels, various tissue cells of body fluids and immune cells work. Normally, the inflammatory response induced by external invading bacteria is a defense system for protecting the living body, whereas abnormally excessive inflammatory response is induced, and various diseases appear. These diseases are called inflammatory diseases. The inflammatory disease is a disease that threatens human life by amplifying and sustaining inflammation of various inflammatory mediators secreted from target cells activated by external stimuli, such as chronic inflammatory diseases in joints such as acute inflammation and rheumatoid arthritis. have.

The key inflammatory mediators that induce these inflammatory diseases are prostaglandins and hydroxy fatty acids, which are precursors of arachidone by cyclooxygenase (COX) and lipoxygenase. It is produced from arachidonic acid.

However, due to westernized diet, lack of exercise, and environmental pollution, various acute and chronic inflammatory diseases are gradually increasing in modern people. Antibiotics and anti-inflammatory drugs are used as treatments for various inflammatory diseases, and various side effects of drugs are gradually becoming a problem. In order to treat inflammation, various anti-inflammatory substances have been developed, and anti-inflammatory substances reported to date are substances that inhibit the biosynthesis of prostaglandins that are activated in damaged tissue cells and cells involved in inflammation in acute inflammation. These substances have a therapeutic effect on acute inflammation, but only alleviate the primary inflammation for chronic inflammation such as rheumatoid arthritis, and can not expect immunological therapeutic effects and also has various side effects including cardiovascular system.

Therefore, while studying the natural plant-derived plant extract that can be used safely without any problems such as the various side effects and resistance as described above, it is NF-κB, cyclooxygenase-2 (Cyclooxygenase-2, Hereinafter referred to as 'COX-2', by inhibiting the activity of chondroitinase (chondroitinase) has been found to have an anti-inflammatory effect to complete the present invention.

The present invention is an algae extract having anti-inflammatory effect by inhibiting NF-κB, cyclooxygenase-2, Cyclooxygenase-2, chondroitinase and anti-inflammatory composition containing the same as an active ingredient, fermented milk, An object of the present invention is to provide a beverage and a dietary supplement.

In order to achieve the above object, the present invention is a heterogeneous grass extract having an anti-inflammatory effect by inhibiting NF-κB, cyclooxygenase-2, chondroitinase (chondrointinase) as an active ingredient It is characterized by providing an anti-inflammatory composition containing, fermented milk, beverages and health functional food.

Hereinafter, the present invention will be described in detail.

Geranium nepalense sub sp. Thunbergii is a genus belonging to the rat handiaceae family and grows in mountains and fields of Korea, Japan and Taiwan, and its height is about 50cm. Roots do not have straight roots, split into several, stems come out, grow obliquely, and hairs spread out. Leaves are opposite each other, split into 3 ~ 5 pieces, 3 ~ 7cm wide, and often have black pattern. A forked piece is an upside down egg, with a dull, shallow tip, with irregular teeth on the top. Contains large amounts of tannins and quercetin, which is anti-inflammatory, hemostatic, astringent, and bactericidal. In civilian, it is used as a medicine for colon Qatar, dysentery, gastric ulcer, duodenal ulcer, etc. In oriental medicine, Hyuncho (현 草) is used as an antidiarrheal agent, but anti-inflammatory mechanism and anti-inflammatory activity are not known.

Looking in detail the process of efficiently separating the heterogeneous grass extract having an anti-inflammatory effect as follows.

In the case of hot water extraction, the weight of the heterozygous sample is measured, the sample is pulverized, 10 times distilled water is added, extracted at 110 ° C. for 3 hours, filtered twice with Whatman filter paper, concentrated using a distiller, and lyophilized. The freeze-dried alien sample is weighed, dissolved by adding an appropriate amount of distilled water, filtered through a 0.2 μm filter and stored in a freezer until use.

In the case of methanol extraction, weigh the samples of the heterozygous grass, grind the sample, add methanol with a purity of 99.9% or more of 5 times the sample weight, and store it for 72 hours at room temperature to induce extraction. After filtration and concentration in a distiller, two times the volume of the distilled water was added to the distilled water, frozen and lyophilized. The freeze-dried samples were weighed, and an appropriate amount of DMSO was added to dissolve the extract. Store in freezer.

The anti-inflammatory pharmaceutical composition containing an extract of J. coli having an anti-inflammatory effect according to the present invention as an active ingredient may be administered through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular. In addition, the anti-inflammatory pharmaceutical composition containing a heterogeneous grass extract having an anti-inflammatory effect according to the present invention as an active ingredient can be formulated into various formulations, when formulated, fillers, extenders, binders, wetting agents, It may be formulated using diluents or excipients such as disintegrants and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which include at least one excipient (eg, starch, sucrose, lactose and gelatin), etc. It is mixed and prepared. Lubricants may also be used in addition to simple excipients. Liquid preparations for oral administration include suspensions, solvents, emulsions, and syrups. In addition to the commonly used simple diluents, water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives, etc. May be included. Formulations for parenteral administration include sterile aqueous solutions, water-insoluble solvents, suspensions, emulsions, lyophilized formulations and suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. Glycerol and gelatin may be used as the base of the suppository.

For humans, a typical daily dose may range from 1 to 30 mg / kg body weight and may be administered once or in divided doses. However, the actual dosage should be determined in light of several relevant factors such as route of administration, age, sex and weight, and health status.

Yield grass extract of the present invention is a preparation that can be used safely in long-term use for the purpose of preventing or treating inflammation because there is almost no toxicity and side effects as a raw material used for food or medicinal herbs.

On the other hand, the present invention can be used as an anti-inflammatory fermented milk, beverages and health functional foods containing the hot water extract of alien grass obtained by the extraction method as an active ingredient.

The anti-inflammatory fermented milk containing the hot water extract of the algae pool as an active ingredient is lactobacillus casei HY2782 { Lactobacillus casei HY2782; Deposited Organization: Korean spawn association, Accession No. KFCC-10813, Date of Deposit, December 7, 1993, Patent Registration No. 0125219 (Name of invention: Lactobacillus skazy HY2782 and product using it) Dissolve juice concentrates, dietary fiber, glucose, oligosaccharides, calcium, vitamins, hot water extracts of heterozygous syrup in a culture solution, mix and stir in a proportion, and homogenize and prepare fermented milk.

In addition, the anti-inflammatory drink containing the hot water extract of the algae pool as an active ingredient, in addition to containing the hot water extract of the algae pool as an active ingredient, added to the calcium bottle, concentrated extracts, liquid fructose, purified water and mixed into the drink bottle sterilization After cooling to room temperature to prepare a beverage.

In addition, the health functional food for inflammation prevention containing the hot water extract of the algae pool as an active ingredient is a nutritional supplement component (vitamin B1, B2, B5, B6, E and acetate ester, nicotinic acid amide), oligosaccharide, 50 After mixing and adding% ethanol and purified water to form granules and drying them in a vacuum dryer, the granules are uniformly prepared by passing through 12 to 14 mesh and extruded by appropriate amounts to form tablets or powders or filled into hard capsules. To produce a hard capsule product.

As described above, the heterozygous extract of the present invention has an excellent anti-inflammatory effect by inhibiting NF-κB, cyclooxygenase-2 and chondroitinase, thereby treating inflammatory diseases. And pharmaceutical compositions for the purpose of prevention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are not intended to limit the scope of the present invention, it is possible for the ordinary change by those skilled in the art within the scope of the technical idea of the present invention.

<Example 1>

Separation and Purification of Algae Extract

Hydrothermal extract of algae grass

After purchase, the water was dried and washed with air, weighed, and ground at intervals of 1 cm in length. 1 kg of the pulverized sample was placed in an extraction bottle, and 10L of distilled water was added thereto, followed by extraction at 110 ° C. for 3 hours. The filtrate by the filter paper was concentrated under reduced pressure using a vacuum rotary concentrator (Eyela NE, Japan), and then lyophilized to prepare a hot water extract of the heterogeneous grass.

Weighing hot water extract of the heterozygous pool was dissolved by adding an appropriate amount of distilled water and filtered through a 0.2㎛ filter and stored in the freezer until use. 10 mg of the dried sample was dissolved in purified water at a concentration of 1 ml and used as an anti-inflammatory efficacy measurement material.

Methanol extract of algae grass

After purchase, the water was dried and washed with air, weighed, and ground at intervals of 1 cm in length. 1 kg of the pulverized sample was placed in an extraction bottle, 5 L of methanol having a purity of 99.9% or more was added thereto, followed by extraction for 72 hours at room temperature, followed by primary filtration with 60 mesh and secondary filtration with filter paper (Whatman No. 41). The filtrate by the filter paper was concentrated under reduced pressure using a vacuum rotary concentrator (Eyela NE, Japan), and then distilled water of 2 times the concentration of the concentrate was added, frozen, and freeze-dried to prepare a methanol extract of the coriander.

10 mg of the dried sample was dissolved in DMSO at a concentration of 1 ml and used as an anti-inflammatory efficacy measurement material.

<Example 2>

Preparation of pharmaceutical composition using extract

It describes an example of the formulation of the pharmaceutical composition containing the extract of the haricot grass of the present invention as an active ingredient, but the present invention is not intended to limit it but only to explain in detail.

Preparation of Capsules

A capsule was prepared by thoroughly mixing 100 mg of Helicobula extract sample, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate, and then filling the hard gelatin capsule according to a conventional method of preparing a capsule.

Manufacture of powder

After mixing 2g of algae extract extract and 1g of lactose completely, it was filled into an airtight cloth to prepare a powder according to a conventional powder preparation method.

<Example 3>

Fermented Milk Manufacture Using Hot Water Extract

Crude milk was sterilized for 15 seconds at 72 ~ 75 ℃ to adjust the nonfat milk solid content to 8 to 20% by weight. The sterilized crude oil was cooled to 37 ° C., and then inoculated with Lactobacillus casei HY 2782 at a concentration of 10 ⁶ cfu / ml, and then cultured and cooled at 37 ° C. until the culture solution had a pH of 4-5. Syrup contains 0.1-50% by weight of juice concentrate, 0.1-20% by weight of dietary fiber, 0.5-30% by weight of glucose, 0.1-15% by weight of oligosaccharides, 0.001-10% by weight of calcium, 0.0001-5% by weight of vitamins and 0.01 After dissolving ~ 5% by weight, sterilized and cooled. The syrup and the culture solution thus prepared were mixed and stirred at a volume ratio of 1: 3, homogenized, and then packaged in a container to prepare fermented milk. The produced fermented milk showed good results in flavor, physical properties and overall taste.

<Example 4>

Beverage Preparation Using Lichen Extract Hot Water Extract

Mix 14% by weight of hot water extract of Heteroasperm extract, 1.5% by weight of jujube extract, and 7% by weight of Angelica Concentrate, and add 7.4% by weight of liquid fructose, add purified water, mix and fill into a drink bottle and sterilize at 90 ~ 95 ℃ Cooled to produce a beverage.

<Example 5>

Preparation of Health Functional Food Using Hot Water Extract

Nutritional supplements (vitamins B1, B2, B5, B6, E and acetic acid ester, nicotinic acid amide), oligosaccharides, and 50% ethanol in 0.01 to 30% by weight, It was added to the weight percent and mixed in a high speed mixer. 10% by weight of sterile purified water was added to the mixture, mixed, and molded into granules having a diameter of 1 to 2 mm. The molded granules were dried in a vacuum dryer at 40 ° C.-50 ° C., and then uniformly prepared granules were passed through 12 to 14 mesh. The granules prepared as described above may be extruded by appropriate amounts into tablets or powders or filled into hard capsules to produce hard capsule products.

<Test Example 1>

NF-κB Inhibitory Activity of Methanol Extract

The methanol extract samples of the heterozygous grass prepared in Example 1 were used to determine the inhibitory activity of NF-κB, which plays a major role in the mechanism of inflammation. For this, Caco-2 cell (ATCC HTB-37), a human colon cell-derived cell line, and three NF-κB transcriptional regulators are present in succession, followed by luciferase gene as a reporter gene. 3κB, a plasmid vector designed to express luciferase upon activation of κB was used.

As a test method, transfection was performed using 2.5 μg of 3κB plasmid and 6.25 μl of lipofectamine 2000 (Invitrogen, Inc.) on Caco-2 cells grown about 50% in T25 flasks. After 6 hours, the cells were collected and transferred to a 96 well plate, simultaneously treated with 5 ng / ml of Interleukin-1β and IL-1β, which were used to activate NF-κB, and methanol extract samples of 100 μg / ml After 16 hours, the luciferase reaction was induced using a Bright-Glo ^™ Luciferase Assay System (Promega) kit, and the reaction value was measured using a luminometer (Biotek, Inc.) and the inhibition rate of NF-κB was investigated.

As a result, Methanol extract of Helicoptera showed 105% NF-κB inhibitory activity, which lowered the NF-κB activation of IL-1β to almost the basal level.

<Test Example 2>

Determination of NF-κB Inhibitory Activity by Concentration of Hot Water Extract

The NF-κB inhibitory activity according to the concentration of the heterozygous hydrothermal extract was investigated by luciferase reporter assay using Caco-2 cells and 3κB plasmid vector as in Test Example 1. The results are shown in FIG.

As can be seen in Figure 1, NF-κB inhibitory activity was increased as the concentration of the heterozygous hydrothermal extract was increased, and the IC ₅₀ of the heterozygous hydrothermal extract was about 41 µg / ml.

<Test Example 3>

Of algae hot water extract In vitro  Determination of COX-2 Enzyme Inhibitory Activity

The hydrothermal extracts of the algae grass were used to investigate the effects of COX-2 enzymes, which play an important role in the inflammatory response. COX-2 enzyme inhibitory activity was measured using absorbance. Specifically, 50 nM of COX-2 was added to 100 mM Tris-HCl buffer solution (pH 8.0) at a final concentration, and 0.5 μM of Heme was added thereto. The COX-2 solution and heterogeneous hot water or methanol extract to be measured were mixed by concentration and maintained for 5 minutes. The reaction was started by adding 100 μM of arachidonic acid and 10 μM of TMPD ( N, N, N ', N'-tetramethyl-p-phenylenediamine). After 5 minutes, the absorbance was measured at 590 nm. The results are shown in FIG.

Inhibition rate calculation

    C1: Absorbance of the positive control (test group with COX-2)

    C2: Absorbance of negative control group (test group without COX-2)

    S1: absorbance of sample group (test group with both COX-2 and sample)

    S2: Absorbance of the sample control group (test group without sample without COX-2)

As can be seen in Figure 2, the COX-2 enzyme inhibitory activity increases as the concentration of the heterozygous hydrothermal extract increases, and the IC ₅₀ of the heterohydrothermal hydrothermal extract was 31 μg / ml.

<Test Example 4>

Determination of COX-2 Promoter Inhibitory Activity of Hot Water Extracts

The effects of the control of the expression of COX-2 promoter of Helicoptera hot-water extract were investigated. The inhibitory activity of COX-2 promoter is luciferase when COX-2 is activated by the presence of luciferase gene as Caco-2 cell (ATCC HTB-37) and COX-2 promoter and reporter gene. Was measured using a plasmid designed to be expressed. As a test method, transfection was performed using 2.5 μg of plasmid and 6.25 μl of lipofectamine 2000 (Invitrogen, Inc.) on Caco-2 cells grown about 50% in T25 flasks. After 6 hours, the cells were collected and transferred to a 96 well plate, simultaneously treated with 5ng / ml IL-1β and 100 ㎍ / ml heterozygous hydrothermal extract sample to activate NF-κB and again after 16 hours, the Bright-Glo ^TM Luciferase Assay System The promega kit was used to induce the luciferase reaction, and the reaction value was measured by a luminometer (Biotek) and the inhibition rate of the COX-2 promoter was investigated. The results are shown in FIG.

As can be seen in Figure 3, as the concentration of the extract of hot water extract increased the inhibition rate of the COX-2 promoter and the IC ₅₀ of the hot water extract was found to be 54 μg / ㎖.

<Test Example 5>

Determination of Chondroitinase Enzyme Inhibitory Activity Using Hot Water Extracts

Glycosaminoglycan (GAG) is present in the mucous region of normal gastrointestinal tissues, and the composition of glycosaminoglycans is different in inflammatory colitis patients compared to normal people. Chondroitin and dermatin are one of the major constituents of glycosaminoglycans. Therefore, it is possible to prevent or alleviate colitis by inhibiting the activity of chondroitinase, a chondroitin degrading enzyme.

In addition, Soll et al. Confirmed the alleviation of this inflammation by artificially injecting chondroitin sulfate from joints of humans and animals from trauma such as inflammation (US Pat. No. 5,498,606). Therefore, the presence or absence of inhibitory activity of chondroitinase in vitro was investigated.

Chondroitinase activity was measured using absorbance. Specifically, 25 mU / mL chondroitinase (Sigma) was added to 50 mM Tris HCl buffer solution (60 mM Sodium acetate, pH 8.0), and the heterozygous hydrothermal extract was added by concentration, and maintained at room temperature for 5 minutes. 0.3 mg / mL chondroitin sulfate A (Sigman Co., Ltd.) was added thereto, followed by reaction at room temperature for 30 minutes, and then the reaction solution was reacted with a dimethylmethylene blue (DMB) solution. The absorbance was measured at 525 nm. The results are shown in FIG.

As can be seen in Figure 4, it was found that as the concentration of the hot water extract increased, the inhibitory activity of chondroitinase increased and the IC ₅₀ of the hot water extract was 6 µg / ml.

<Test Example 6>

Cytotoxicity Test of Hot Water Extracts from Algae Grass

MTT assay was carried out using Roche's Cell Proliferation KIT I (MTT) to ^evaluate the effect of Cytophyllae hydrothermal extract on cytotoxicity and cell proliferation in Caco-2 cells. The cell viability assay kit was used for the investigation. The test methods were in compliance with the test methods recommended by each manufacturer. The results are shown in FIGS. 5 and 6.

As can be seen in Figures 5 and 6, when treated with a concentration of 10 to 200 ㎍ / ㎖ hot water extract of the heterogeneous grass was not able to confirm significant toxicity. Therefore, the cytotoxicity of the hot water extract of the algae grass was judged to be safe within the tested concentration range.

1 is a graph showing the results of measuring the NF-κB inhibitory activity according to the concentration of the hot water extract of the genus.

Figure 2 is a graph showing the results of measuring the COX-2 inhibitory effect of each concentration of the hot water extract of the heterogeneous grass.

Figure 3 is a graph showing the results of measuring the inhibitory effect of COX-2 promoter activity for each concentration of the hot water extract of the heterozygous pool.

Figure 4 is a graph showing the results of measuring the chondroitinase (chondroitinase) inhibitory effect of each concentration of the hot water extract of the heterozygous pool.

Figure 5 is a graph showing the results of measuring the cytotoxicity by concentration of the hydrothermal extract of the heterozygous pool by MTT assay.

Figure 6 is a graph showing the results confirmed by the cell proliferation (Vill proliferation) and survival analysis (Viability Assay) of the concentration of the hydrothermal extract of the heterogeneous grass.

Claims (12)

Algae herb extract with anti-inflammatory effect. The method of claim 1, The Yield grass extract is Yield grass extract having anti-inflammatory effect, characterized in that it inhibits NF-κB. The method of claim 1, The heterogeneous herb extract is a heterogeneous herb extract having anti-inflammatory efficacy, characterized in that it inhibits cyclooxygenase-2 (Cyclooxygenase-2). The method of claim 1, The heterogeneous herb extract has an anti-inflammatory effect, characterized in that it inhibits chondroitinase (chondrointinase). The pharmaceutical composition for anti-inflammatory, characterized in that containing the extract of Heilweed grass of claim 1. The pharmaceutical composition for anti-inflammatory, characterized in that containing as an active ingredient extract of the heterogeneous grass of any one of claims 2 to 4. Anti-inflammatory fermented milk, characterized in that it contains a hot water extract of the heterogeneous grass of claim 1 as an active ingredient. Anti-inflammatory fermented milk, characterized in that it contains a hot water extract of the heterogeneous grass of any one of claims 2 to 4 as an active ingredient. Inflammation-preventing drink, characterized in that it contains a hot water extract of the heterogeneous grass of claim 1 as an active ingredient. Inflammation-preventing drink, characterized in that it contains a hot water extract of the heterogeneous grass of any one of claims 2 to 4 as an active ingredient. Anti-inflammatory health functional food, comprising the hot water extract of the heterogeneous grass of claim 1 as an active ingredient. Claims 2 to 4 of the functional food for preventing inflammation, characterized in that it contains a hot water extract of the heterogeneous grass of any one of the claims as an active ingredient.

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